The long term objectives are to provide a molecular understanding of ion permeation and voltage-dependent conformational changes (gating) for ion channels in general and potassium channels in particular. Inward rectifier potassium channels (IRKs) are used as the targets for achieving such understanding. IRKs are important because they regulate resting potential and the shape of action potentials. In turn, they may be regulated by a number of important physiological ligands such as G proteins, ATP and phosphorylating kinases. IRKs have the advantage of a simpler structure than most voltage dependent ion channels. The specific aims are to: 1) locate the pore and gating domains of IRKs and analyze their mechanisms; 2) identify molecules that may act as physiological blockers of IRKs; and 3) determine the biochemical structure of IRKs. The research design makes use of two cloned IRKs, one of which rectifies strongly and the other weakly. The structures responsible for the differences in rectification are localized and analyzed using a combination of mutagenesis, heterologous expression, and electrophysiology. Rectification involves cytoplasmic molecules such as Mg2+_ and may also involve naturally occurring polyamines such as spermine or spermidine. Whether the polyamine effects are physiological will be tested by biochemical and electrophysiological methods. More direct structural data on IRKs may be obtained from analysis of purified protein. There is no rich source of IRK protein so we will use overexpression followed by immunopurification to obtain sufficient quantities. Topological models of protein folding will be tested using glycosylation site insertion. By determining the mechanisms of ion permeation and voltage-dependent gating in IRKs we may gain a more general understanding of these mechanisms in voltage-dependent ion channels. Ion channels are the essential molecules of excitability which is the fundamental property of the nervous system and a basic property of living cells. Voltage-dependent ion channels may be involved in and are targets for treatment of diseases of the nervous system such as Alzheimer's disease and epilepsy. Achieving our objectives will help to provide a more rational basis for the therapy of these diseases.